The Shc protein helps to transmit signals from receptor andcytoplasmic tyrosine kinases to Ras. We have shown that severalbreast cancer cell lines (MDA-MB-453, BT-474, MDA-MB-361, andSKBR3), which overexpress the ErbB2 receptor tyrosine kinase,contain constitutively tyrosine phosphorylated Shc. To investigatethe role of Shc in these cells, we transfected them with a Shc-Y317Fdominant-negative mutant defective in signaling to Ras. Thetransfectants were unable to form stable colonies, suggestinga critical role for Shc in the proliferation of these cells.In contrast, dominant-negative Shc transfectants of the nontransformedbreast epithelial cell line HBL-100 grew normally. Surprisingly,cell cycle analysis of transfected SKBR3 cells suggested thatthe cells were blocked not only in G0-G1, but also in G2-M.The G2-M block was unexpected because Shc-Y317 is downstreamof receptor tyrosine kinases that drive the early events inthe cell cycle. Both the G0-G1 and G2-M arrest were rescuedby transfection with wild-type Shc or oncogenic Ras 12V. Rescueby Ras suggests that Shc Y317 signals upstream of Ras, and thatShc to Ras effector pathways are involved in G2-M, althoughconfirmation awaits a detailed molecular analysis. Most importantly,this work provides the first evidence for Shc involvement inG2-M.

We have focused our attention on the role in breast cancer ofthe adapter protein, Shc. Activated ErbB2 and other growth factorreceptors phosphorylate Shc on tyrosine 317, which in turn isrecognized by the SH2 domain of Grb2, typically found complexedwith SOS (reviewed in Refs. 19
and 20
). As a result, the complexesare translocated to the cellular membrane, where SOS may facilitateGDP release from Ras, thereby allowing Ras to bind GTP and returnto its active state (32, 33, 34)
. Indeed, signaling pathwaysto Ras appear activated in some breast cancer cells (8, 15, 35,36, 37, 38, 39, 40)
. In this regard, Janes et al.(15)have reported that Grb2 and SOS are constitutively associatedwith ErbB2 in the breast cancer cell lines, SKBR3 and BT474,and we have reported recently that several breast cancer celllines that overexpress ErbB2 contain tyrosine-phosphorylatedShc, complexed to Grb2 (35)
. However, Grb2 can dock to growthfactor receptors by at least two different mechanisms: directlythrough the SH2 domain of Grb2, and indirectly through dockingto tyrosine phosphorylated Shc, which through its own SH2 domainis docked to other phosphorylated tyrosines in the receptor(19, 33, 41)
.

Therefore, we transfected ErbB2-positive breast cancer celllines and the nontransformed breast epithelial cell line, HBL-100,with either the Shc-Y317F mutant contained in a pEBG vectoror the pEBG vector alone as a control, and cotransfected allof these cells with pNeoNut to confer G418 resistance. The Shc-Y317Fmutant profoundly inhibited the ability of breast cancer cellsto form colonies (Fig. 1
and Table 1
). The colonies were bothdramatically smaller (Fig. 1A)
and fewer in number than coloniesfrom cells transfected with the vector alone (Fig. 1B
and Table1
). Only 3% of cells in the mutant colonies incorporated BrdUcompared with >40% of the cells from the parental colonies(Fig. 1C)
, suggesting that the mutant Shc was inhibiting theability of the breast cancer cells to proliferate. In supportof this view, the TUNEL assay and cell morphology suggestedcomparably low levels of apoptosis (24%) in cells fromboth the mutant and parental colonies (data not shown). In contrastto the growth inhibition seen in the breast cancer cells, thenontransformed breast HBL-100 cells transfected with the Shc-Y317Fmutant formed colonies equal in both size and number to thevector alone controls (Fig. 1B
and Table 1
). We attemptedto derive stable lines from SKBR3 Shc-Y317F transfectants byadding exogenous growth factors to the individual microcolonies,subcultured in 96-well plates. However, the colonies failedto grow in response to either EGF (30ng/ml and 5ng/ml), insulin(30 µg/ml and 5 µg/ml), lysophosphatidic acid (10µM), 20% serum, or transferrin (5 µg/ml) (data notshown). The results of the colony-forming assays indicate thatthe Shc dominant negative was able to suppress proliferationin several ErbB2-positive cell lines and further imply thatShc interactions with Grb2 are necessary for the proliferationof the ErbB2-positive breast cancer cells but not for proliferationof the nontransformed breast epithelial cells.

Fig. 1. Shc dominant-negative mutant, Shc-Y317F, inhibits colony formation in several breast cancer cell lines. A, colonies formed by SKBR3 cells transfected with the dominant-negative Shc mutant are greatly reduced in size. Photomicrographs of colonies obtained from: SKBR3 cancer cells that had transfected with the empty vector (SKBR3-pEBG) or dominant-negative Shc (SKBR3-pEBG-ShcY317F). x30. B, comparison of colony-forming ability of cells transfected with either the pEBG empty vector () or the dominant-negative Shc (). The assay of colony formation is described in Table 1
. Results are expressed as a percentage of colonies formed by cells transfected pEBG-Shc-Y317F compared with colonies transfected with vector alone. Bars, SD of three separate experiments. *, colony numbers statistically different (P < 0.001) by Students t test from cells transfected with the empty vector. C, Shc dominant-negative inhibits DNA synthesis in SKBR3 cells. SKBR3 cells were transfected with the indicated constructs and then cultured for 3 weeks under G418 selective pressure. Multiple colonies were harvested, pooled, and tested for their ability to incorporate BrdU. Panels show phase contrast photomicrographs of cells using bright-field illumination (left panels) or immunofluorescent staining nuclei that have incorporated BrdU (right panels). Arrow, the same cell in both panels, for orientation. The fields shown are representative of two separate experiments in which 88/210 SKBR3 cells transfected with the empty pEBG vector were scored as positive, whereas only 2 of 63 SKBR3 cells transfected with the pEBG-Shc-Y317F mutant were scored as positive for BrdU incorporation.

Stable Expression of Shc-Y317F in Nontransformed HBL-100 Mammary Epithelial Cells.
Unlike the Shc-Y317F transfectants of the breast cancer celllines, sufficient numbers of cells from Shc-Y317F transfectedHBL-100s could easily be obtained to permit analysis of Shcexpression by immunoblotting. The Mr 80,000 Shc-Y317F-GST fusionprotein was highly expressed in several expanded clones, insome cases at levels comparable with the expression of Mr40,000,Mr 52,000, and Mr 66,000 endogenous Shc proteins (Fig. 2)
.Expression of the mutant protein did not diminish, even afterseveral passages in culture (data not shown). Thus, unlike SKBR3and the other ErbB2-positive breast cancer cells, the nontransformed,HBL-100 cells do not appear to depend upon signaling from Shc-Y317for proliferation.

Fig. 2. Expression of the Shc-Y317F-GST fusion protein in stable HBL-100 transfectants. Cells were transfected with pEBG-Shc-Y317F using Lipofectin and then cultured for 3 weeks under G418 selective pressure. Cell extracts of G418-resistant clones (named AMT through VMT) were immunoprecipitated using anti-Shc polyclonal antibodies cross-linked to protein A-Sepharose beads, and the proteins were resolved on an SDS 7.5% polyacrylamide gel, blotted, and probed with anti-Shc antibodies as detailed in "Materials and Methods." *, location of the Mr 80,000 Shc-GST fusion protein; arrows, location the endogenous Shc isoforms.

Cell Cycle Changes Caused by Shc-Y317F.
Efforts to explore the biological effects of the Shc dominant-negativemutant on the ErbB2 positive breast cancer cells were hamperedby the paucity of cells obtainable from stable transfections(see Fig. 1A
). To circumvent this problem, we chose to examineSKBR3 cells that were transiently expressing the mutant Shcprotein. Accordingly, SKBR3 cells were transfected with eithernormal Shc (pEBG-ShcWT) or mutant Shc (pEBG-Shc-Y317F) and thencultured in nonselective media for up to 82 h. We then analyzedexpression of the Shc-Y317F-GST fusion protein at various times.Expression of both the Mr 80,000 wild type and Shc-Y317F proteinswere optimal 60 h after transfection (Fig. 3)
.

Fig. 3. Expression of Shc-GST fusion protein in transiently transfected SKBR3 cells. Cells were transfected with pEBG-Shc-Y317F (numbered lanes) or pEBG-ShcWT (WT lane) using Lipofectin, cultured up to 84 h, and then analyzed for expression of Shc proteins as in Fig. 2
.

Shc is known to be important in the signaling of several growthfactor receptors to Ras (42, 43, 44, 45, 46, 47, 48, 49)
. Becauseof the well-known involvement of Ras in passage through theG1 phase of the cell cycle (28, 29, 30, 31)
, we hypothesizedthat the dominant-negative Shc was inhibiting colony formationby the SKBR3 cells and other ErbB2-positive breast cancer cellsby blocking passage through G1. To test this possibility, wetransiently cotransfected wild-type Shc or Shc-Y317F togetherwith the transfection marker, pGreen LanternTM. Transfectedcells (typically 1020% transfection efficiency) wereidentified by expression of the green fluorescent protein, whichcould be detected as early as 24 h after transfection by fluorescencemicroscopy (data not shown) or flow cytometry (Fig. 4)
. Bystaining cellular DNA with propidium iodide and instructingthe flow cytometer to score only green fluorescent protein-positivecells, we were able to determine the cell cycle distributionof cells that were presumably coexpressing the Shc-GST fusionproteins. Analysis of DNA histograms indicated that neitherwild-type Shc nor the pEBG vector alone affected the proportionof SKBR3 cells in G0-G1, S, and G2-M (Fig. 4, A, B, and D)
.This suggested that expression of the GST-fusion protein didnot disrupt cell cycle progression. In contrast, as predictedby the colony formation assay, Shc-Y317F did appear to inhibitcell cycle progression of the SKBR3 cells (Fig. 4C)
. To oursurprise, however, the cells appeared to be partially blockedin G2-M (an increase from 12 to 24% of cells in G2-M). Thissuggested that Shc may have a critical but previously unrecognizedrole in G2-M progression.

Fig. 4. Cell cycle analysis of SKBR3 cells expressing Shc-Y317F. A, DNA histogram of asynchronously growing, untransfected SKBR3 cells; BE, DNA histograms of SKBR3 cells that had been transfected as above with both 1 µg of pGreen LanternTM and 10 µg of the indicated construct(s) and then harvested 60 h later, stained with propidium iodide, and analyzed by a FACScan® flow cytometer, as detailed in "Materials and Methods." F, DNA histogram of SKBR3 cells exposed for 24 h to 60 ng/ml Taxol, known to arrest cells in G2. Jagged curve, the actual number of cells per channel; smooth curve, the best fit calculated by MODFIT software (Becton Dickinson). By deconvolutional analysis, Modfit then calculated the percentage of cells in each phase of the cell (shaded, labeled areas).

However, Shc-Y317F conceivably could have effects on cellularpathways that are not normally regulated by Shc. In one approachto address this concern, cotransfecting wild-type Shc alongwith Shc-Y317F rescued the arrested phenotype; the cotransfectedcells progressed normally through the cell cycle (Fig. 4E)
.

Although not dramatic, cell cycle analysis of the SKBR3 cellsthat were transiently expressing mutant Shc hinted that Shcis involved in G2-M passage. To address this more definitively,we transfected large numbers of SKBR3 cells (8 x 106) with Shc-Y317Fand the neo selectable marker, and after 3 weeks of culturewith G418, we enumerated the colonies. As we observed previously(Fig. 1)
, colonies from SKBR3 cells transfected with Shc-Y317Fwere greatly reduced in size (data not shown) and number (Fig.5)
, compared with colonies from SKBR3 cells transfected withthe pEBG empty vector. With this large-scale transfection, wewere able to harvest sufficient numbers of the microcoloniesto obtain a pool of 1 x 105 Shc-Y317F cells for cell cycle analysis(Fig. 6AD)
. DNA histograms show clearly that the Shc-Y317Fcells are blocked in both G1 and G2-M. SKBR3 cells transfectedwith the mutant Shc appear only in the G0-G1 (61%) and the G2-M(39%) phases of the cell cycle (Fig. 6D)
, whereas cells transfectedwith the empty vector are found in all three phases of the cellcycle (63% in G0-G1; 28% in S phase; and 9% in G2-M). The BrdUincorporation studies (Fig. 1C)
are quite consistent with theflow data. Thus, the cells transfected with the mutant Shc arenot synthesizing DNA, and therefore their cell cycles are clearlyarrested.

Fig. 5. Oncogenic Ras 12V overcomes the ability of the Shc mutant to inhibit SKBR3 colony formation. Colony formation assays were performed as described in "Materials and Methods." Results are expressed as percentages of colonies formed by cells transfected with the empty vector. Bars, SD.

Fig. 6. Cell cycle analysis of colonies obtained from large-scale transfections of SKBR3. SKBR3 cells were transfected with the indicated construct and then were cultured for 4 weeks under G418 selective pressure. Multiple G418-resistant colonies were harvested and pooled, and cells were stained with propidium iodide similarly to Fig. 4
for analysis by flow cytometry.

Shc Is Phosphorylated on Tyrosine in SKBR3 Cells Arrested in G2 by Taxol or Nocodazole.
If the SKBR3 cells require Shc for passage through G2-M, thenwe might expect it to be tyrosine phosphorylated in these laterstages of the cell cycle. To test this prediction, we arrestedSKBR3 in G2 using Taxol (55)
or nocodazole (56)
and then examinedShc for tyrosine phosphorylation. SKBR3 cells arrested in G2by either taxol or nocodazole showed very high levels of p52-Shctyrosine phosphorylation, even higher than observed in asynchronouslyproliferating SKBR3 cells (Fig. 7)
.

Fig. 7. Shc tyrosine phosphorylation in SKBR3 cells arrested in G2 by Taxol or nocodazole. Cells were cultured in 10% FBS-supplemented culture media with 0.06 µg/ml Taxol (T) for 20 h, with 0.5 µg/ml nocodazole (N) for 14 h, or with no drug (-), and then analyzed for expression of proteins as in Fig. 2
, but first probing with the 4G10 monoclonal antibody to phosphotyrosine, then stripping and reprobing with antibody to Shc protein. A very light exposure of the anti-PY blot is shown so that the increment caused by Taxol and nocodazole can be appreciated.

Active Ras Rescues the Shc-Y317F-induced G1 and G2-M Arrest.
As mentioned above, considerable evidence indicates that Shctyrosine 317 signals through Grb2-SOS to p21Ras, and that activeRas is necessary for passage through the G1 phase of the cellcycle. Accordingly, we predicted that the Shc dominant negativeblock of G1 could be bypassed if the cells contained activeRas. Thus, although we expected that SKBR3 cells cotransfectedwith Shc-Y317F and active Ras would still form only a few, smallcolonies as seen before with Shc-Y317F alone, we did expect,however, that cell cycle analysis would show these double transfectantsto be arrested only in G2-M. We were surprised, therefore, whenSKBR3 cells that were cotransfected with Shc-Y317F and Ras-G12Vformed stable colonies that were equal in both size (data notshown) and number (Fig. 5)
to colonies formed by SKBR3 cellstransfected with Ras-G12V alone. We did observe a 20% reductionin the total number of colonies formed by the Ras-G12V transfectedcells compared with the pEBG vector controls. However, thismay be an artifact of the pUC expression vector that containsRas-G12V, inasmuch as the pUC empty vector controls produced20% fewer colonies as well.

If rescue by Ras-G12V were truly bypassing the need for Shc,then we would expect that Shc-Y317F should still be expressedin the rescued cells. To test this, we analyzed lysates fromthe Shc-Y317F:Ras-G12V clones for the presence of Shc proteins.Anti-Shc immunoblotting demonstrated that the Mr 80,000 ShcTM-Y317F GST fusion protein was expressed in these cells, althoughat a lower level than the endogenous Shc proteins (Fig. 8)
.

Fig. 8. Expression of the Shc dominant negative in SKBR3 cells cotransfected with Ras-12V. Cells were transfected by Lipofectin (6 µl/ml) and cultured in complete medium under G418 selective pressure for 4 weeks. Detergent lysates of cells were clarified, mixed with Laemmli sample buffer, and resolved on a SDS 7.5% polyacrylamide gel as described previously. Proteins were blotted and probed with antibody to Shc. The molecular weight of the Shc-GST fusion protein is Mr 80,000, as indicated by the *. Arrows on the left, endogenous Shc isoforms.

The surprising ability of Ras-G12V to overcome the Shc-Y317Fblock in colony formation suggested that the Shc mutant functionedupstream of Ras-G12V both in G0-G1 and in G2-M. Consistent withthis notion, cell cycle analysis of colonies from the Ras-G12V-rescuedSKBR3 Shc-Y317F transfectants revealed that Ras-G12V rescuednot only the G0-G1 but also the G2 arrest (Fig. 6F)
.

We also observed an increase in dead cells in the "stable" transfectants(see particularly Fig. 6, CF
, the shaded area of thegraph between 0 and 10 on the abscissa) compared with the SKBR3cells transiently expressing the various recombinant proteins(see Fig. 4
). This may be because the stable transfectantshad been fighting for survival for 6 weeks under G418 selectivepressure. The dead cells do not appear related to the expressionof only Shc proteins, because cells transfected with the oncogenicRas showed similar levels of nonviable cells (Fig. 6E)
.

Shc-Y317F: a Highly Dominant-Negative Mutant in ErbB2-overexpressing Breast Cancer Cells.
It is perhaps surprising that no colonies expressing only G418resistance emerged from the neo/Shc-Y317F transfectants. Althoughthe Shc:neo DNA ratio was high (5:1), we might have expectedthat some cells would have stabily incorporated and expressedonly the G418 resistance gene. These data suggest that the Shcmutant is acting in a highly dominant manner and is not merelycompeting with endogenous normal Shc for SH2/PTB binding sites.This dominance is unlikely to be an artifact of the GST-fusionprotein, because not only did expression of GST-WT-Shc not adverselyaffect SKBR3 proliferation, but it also rescued SKBR3 cellsthat were cotransfected with the mutant Shc. Consistent withour results, Baldari et al.(57)
reported that a Shc dominant-negativeconstruct, the Shc-SH2 domain, potently inhibited CD4-mediatedactivation of NF-AT in Jurkat cells (57)
. The mechanism ofthis highly dominant behavior might be better understood ifone could compare: (a) the cellular molecules with which Shc-Y317Fand normal Shc associate; and (b) the subcellular distributionsof the normal and mutant Shc proteins.

Unlike SKBR3 cells, proliferation of the nontransformed breastepithelial cell line HBL-100 was clearly unaffected by the presenceof SHC-Y317F (Figs 1
and 2
). Shc-Y317F transfectants of HBL-100cells formed colonies equal in size and number to HBL-100 cellsthat had been transfected with just the empty vector (Fig. 1)
.This suggests either that HBL-100 cells do not require Shc signaling,or that they are otherwise much less sensitive to interferenceby the mutant Shc. Inasmuch as autocrine fibroblast growth factor-2reportedly drives HBL-100 proliferation (58)
, fibroblast growthfactor receptor signaling may be less dependent than ErbB2 signalingon Shc (59, 60, 61, 62, 63)
. Alternatively, the SKBR3 and HBL-100cells may differ in other signaling components or in regulatorsof the cell cycle.

Shc Is the Major Ras-activating Pathway in ErbB2-overexpressing Breast Cancer Cells.
Although Ras activity can be regulated by many mechanisms, oftenRas appears to be activated when Grb2-SOS binds to tyrosine-phosphorylatedgrowth factor receptors and is thereby translocated to the cellularmembrane, where it gains access to Ras (32, 33, 34)
. Grb2-SOScan bind to growth factor receptors either directly by dockingto receptor phosphotyrosines, or indirectly by docking to thetyrosine-phosphorylated 317 residue of Shc (19, 32, 33, 34, 41). On the basis of the ability of Shc-Y317F to inhibit proliferationof ErbB2-overexpressing breast cancer cells and on the abilityof Ras to rescue the SKBR3 cells from Shc-Y317F-induced growtharrest, we conclude that the indirect pathway through Shc toRas is dominant and required for growth of these cells (rigorousproof of this will require a molecular analysis of Shc, itsassociated proteins, and Ras activity in cells expressing Shc-Y317F;such analyses will require substantial numbers of cells expressingthe mutant Shc, most readily accomplished by engineering themutant Shc gene to be under the control of an inducible promoter).This is clearly not the case for all cells. Shc-Y317F does notinhibit the growth, as shown here, of the nontransformed breastepithelial cell line HBL-100 (Fig. 1
and Table 1
). Furthermore,Shc-Y317F does not inhibit the growth of NIH3T3 cells (43, 51)or Rat-1 fibroblasts (53)
in serum-supplemented media.However, Shc-Y317F does inhibit EGF-induced DNA synthesis (43)and serum-free growth and transformation of NIH3T3 cells (51)
.Another dominant-negative Shc construct consisting of only theShc SH2 domain effectively inhibited EGF responses in NIH3T3cells (45)
, in 293T human kidney epithelial cells (64)
, andin rat fibroblasts (46)
. Similarly, the Shc SH2 domain inhibitedPDGF-induced DNA synthesis in NIH3T3 cells (48)
. Further consistentwith a prominent role for Shc in EGF responses, a phosphotyrosyl-peptidethat can bind to the Shc SH2 domain inhibits EGF-driven Rasactivity in permeabilized PC12 cells (42)
.

However, interpretation of the inhibitor studies that used ShcPTB and SH2 domains or the SH2-binding site peptide has recentlybeen complicated by the discovery of several additional Shcsites involved in molecular interactions. These include twoadditional tyrosine phosphorylation sites on Shc, at the vicinalresidues 239 and 240 located in the Shc CH1 domain (43, 44, 54, 65, 66, 67)
. These sites may act in concert with tyrosine317 in binding Grb2, but in addition to binding several as yetunidentified cellular proteins (44, 67, 68)
, appear to driveRas-independent activation of Myc in NIH-3T3 cells respondingto EGF and in BaF3 mouse myeloid cells responding to IL-3 (43, 54). Thus, a dominant-negative Shc construct consisting ofthe Shc SH2 domain (or of the Shc PTB domain) might be not onlya more potent inhibitor of Ras activation than Shc-Y317F butalso an inhibitor of Ras-independent pathways as well. In contrast,we have no reason to suspect that Shc-Y317F interferes withShcY239,240 signaling in breast cancer cells:

(a) In BaF3 cells Shc-Y317F interfered with IL-3 signaling throughShc to Ras but did not interfere with IL-3 survival signalsthrough Shc(Y239,240) to Myc (54)
. Similarly, the Shc-Y317Fmutant did not interfere with Shc signaling through Shc(Y239,240)to Myc in NIH-3T3 cells responding to EGF (43)
.

(b) We were able to rescue the SKBR3 Shc-Y317F cells with oncogenicRas, and therefore do not need to invoke effects of Shc-Y317Fon Ras-independent pathways to explain the effects of the mutanton the growth of the ErbB2-positive breast cancer cells.

Shc, Ras, and the Cell Cycle.
To the extent that Ras is activated via Shc-Y317, we expectedthe Shc-Y317F mutant to inhibit Ras activation. Active Ras isrequired for transit through two early phases of the cell cycle:G0 to G1(28)
and mid/late-G1 to S (29, 30, 31)
, where Rasinduces cyclin D1 and down-regulates the cyclin-dependent kinaseinhibitor, p27KIP1(31)
. Therefore we predicted that SKBR3cells transfected with the Shc-Y317F mutant would, if affectedat all, arrest in G0-G1. Indeed, 60% of the Y317F SKBR3 cellsdid arrest in G0-G1, and this arrest could be rescued by oncogenicRas, as expected (Fig. 6)
.

Unexpectedly, however, nearly 40% of the cells were blockedin G2-M, and this arrest too could be rescued by oncogenic Ras,implying that in SKBR3 cells (and by extension, in the otherErbB2-overexpressing breast cancer cells), Shc signaling toRas is required for G2-M passage. (However, a caveat to therescue experiments is that oncogenic Ras (or WT-Shc) could beaffecting parallel pathways impacting cell growth; or coexpressionof oncogenic Ras could be partially suppressing expression ofthe mutant Shc protein. The latter would be consistent withthe low level of mutant Shc expression relative to endogenousShc in the Ras-rescued cells. Alternatively, the mutant Shcmay be a potent dominant negative. As discussed above, thesesorts of questions could be addressed if the genes were engineeredto be under control of inducible promoters.)

The differences in cell cycle distribution of SKBR3 cells thatwere transiently expressing (Fig. 4)
and stabily expressing(Fig. 6)
the Shc mutant are somewhat puzzling. For example,transiently expressing cells had the normal number of cellsin S phase but fewer in G0-G1 and more in G2-M. In contrast,stabily expressing cells had no measurable cells in S phase,more cells in G2-M, but normal numbers of cells in G0-G1. Alikely explanation derives from the observations that SKBR3cell cycle time is long, 40 h, and in the transiently expressingSKBR3 cells, the mutant Shc protein does not reach maximum expressionuntil somewhere between 2460 h after transfection. Becausethe transiently expressing cells were harvested for analysis60 h after transfection, they would have experienced the effectsof the mutant Shc for less than one full cell cycle. Thus, morepronounced changes would be observed in the number of cellsin the rapid phases of the cell cycle (about 4 h for G2-M inSKBR3) than in the longer phases of the cell cycle (about 24h for G0-G1 and about 12 h for S phase in SKBR3 cells). Additionally,it is possible that G2-M is more sensitive than G1 to low levelsof the mutant Shc.

However, if Shc is activating Ras in G2 through Shc-Grb2-SOScomplexes, we would expect to find Shc tyrosine phosphorylatedin G2. Consistent with this prediction, SKBR3 cells arrestedin G2 by either the microtubule-stabilizing drug, Taxol, orby the microtubule-disrupting agent, nocodazole, contained veryhigh levels of tyrosine-phosphorylated Shc (Fig. 7)
. To date,only a few scattered reports have suggested that Ras activityis required in G2. G2-M passage in A549 lung adenocarcinomacells is blocked by a farnesyl transferase inhibitor, FTI-277,which prevents Ras from localizing properly to the cell membrane(69)
. However, one cannot rule out effects of the inhibitoron other farnesylated proteins, such as the nuclear lamins.More definitively, in elegant experiments using a temperature-sensitiveKi-Ras, normal rat kidney cells grown in the absence of serumwere dependent upon Ras being active during G2 for progressionthrough G2(29)
. Suggesting one mechanism for the Ras G2 requirement,active Ras and Myc cooperate in primary rat embryo fibroblaststo induce cdc2 (70)
, a kinase that complexes with cyclins Aand B and is well known to be required for G2 progression (71, 72).

A second potential link between Shc and passage from G2 to Mis suggested by recent findings that chicken embryo fibroblaststhat had been transformed with v-erb-B contained complexes oftyrosine-phosphorylated Shc and caldesmon colocalized in thecells to areas of actin stress fiber disassembly (73)
. Stressfiber disassembly is necessary to permit the rounding-up ofcells prior to mitosis and is required for normal passage ofadherent cells through the G2-M boundary (74, 75)
.

A role for Shc in G2-M raises several questions. What kinaseis activating Shc during G2-M? Several lines of evidence suggestSrc family proteins as candidate kinases of Shc in G2. Src familymembers, Src, Yes, and Fyn, are all activated in NIH3T3 cellsresponding to PDGF and are required for PDGF-driven passagethrough G2-M (48)
. Furthermore, Shc is tyrosine phosphorylatedin cells transformed by oncogenic v-src and in vitro kinaseassays have also shown that Shc can act as a substrate of Srcfamily members (19, 76)
. Particularly relevant here, Src familytyrosine kinases are activated in breast cancers and breastcancer cell lines, including SKBR3 (77, 78, 79)
.

Alternatively, ErbB2 or other growth factor receptors couldbe responsible for the G2 Shc tyrosine phosphorylation in SKBR3cells and the other ErbB2-overexpressing breast cells. Thesebreast cancer cells display various fibroblast growth factorreceptors, as well as receptors for insulin, insulin-like growthfactor-1, EGF, heregulins, Cripto-1, hepatocyte growth factor,mammary-derived growth factor, and others (8, 80, 81, 82)
.In this regard, a tyrosine kinase inhibitor, tyrphostin AG879,reportedly has high specificity for ErbB2 relative to the EGF,PDGF, and nerve growth factor receptors (83)
. AG879 markedlyinhibits both ErbB2 autophosphorylation and Shc tyrosine phosphorylationin asynchronously cycling SKBR3 cells (35)
, suggesting thatat least in these cells, ErbB2 is the primary driver of Shcactivity. However, it is not known to what extent Shc is phosphorylateddirectly by ErbB2 or indirectly by ErbB2 activating other cellularkinases such as Src.

In conclusion, our results imply that signaling from Shc tyrosine317 to Ras is necessary for transit of both the G1 and G2-Mphases of the cell cycle in several ErbB2-positive cell lines.This has important implications for growth control in breastcancer cells and suggests Shc-Y317 as a potential therapeutictarget in human breast cancer.

Colony Formation Assay.
Transfections were carried out in duplicate as mentioned aboveusing equal amounts of DNA for each 100-mm tissue culture plate.SKBR3 cells (1 x 106) were transfected by Lipofectin with thefollowing plasmid combinations: pEBG Wild Type Shc, pEBG, pEBG-Shc-Y317F,pEBG-Shc-Y317F and pUC-Ha-Ras-G12V, and pUC-Ha-Ras-G12V alone.Two days after transfection, cells were split 1:4 and culturedfor 3 weeks in complete medium supplemented with G418 (500 µg/ml).Resistant colonies were counted and harvested for cell cycleanalysis. Data were obtained from at least two independent transfectionassays performed in triplicate.

BrdU Incorporation.
SKBR3 cells (1 x 106) were transfected by Lipofectin with thepEBG and pEBG-Shc-Y317F plasmids, and subjected to G418 selectionpressure for 3 weeks, as described above. Sixteen h before assay,the G418-resistant colonies were harvested, pooled, and seededat 1 x 104 per well on an eight-chambered culture slide. Fourteenh after plating, cells were incubated with BrdU (10 µM;Becton Dickinson) for 2 h and then fixed in 70% cold ETOH for30 min (87)
. After allowing the slides to air dry, the cellswere treated with 0.7 NaOH for 2 min, neutralized in cold 1xPBS, and probed with anti-BrdU (20 µl/chamber; BectonDickinson) in 50 µl of 0.01 MTRIS, 0.14 M NaCl, 0.1% Tween-20,pH 7.6 (TBST) for 1 h at room temperature. This was followedby three washes, 2 min each, in TBST. Secondary antibodies (Goat-anti-mouseIgG, ALEXA conjugated, diluted 1:100) were added to slides for30 min at room temperature. The cells were coverslipped in 10%glycerol mounting media, and positive cells were visualizedwith a fluorescence microscope (AO Ultrastar) using 450495nm bandpass excitation and 520 nm longpass emission filters,along with sufficient white light to just allow visualizationof non-fluorescing cells.

Cell Cycle Analysis.
Asynchronous SKBR3 cells were transiently transfected with 10µg of pEBG Wild Type Shc or 10 µg of pEBG-Shc-Y317Fin the presence of pGreen Lantern (1 µg). The transfectedcells were harvested 60 h after transfection by trypsinization,fixed in 70% cold ethanol for 30 min, washed in PBS, and thenstained with 5 µg/ml propidium iodide, 0.1 mg/ml RNaseA, 0.1% NP40, and 0.1% trisodium citrate for 30 min. Fluorescencewas determined using a FACScan flow cytometer (Becton Dickinson,San Jose, CA); pGreen Lantern fluorescence in the FITC channeland propidium iodide fluorescence in its channel were compensatedfor overlap using cells stained with only one or the other dye.Data were gated to exclude unstained cells and cell debris andwere also gated on the pGreen Lantern fluorescence to includein the analysis of propidium iodide fluorescence only thosecells that were expressing the transgene. Data were analyzedby MODFIT software (Becton Dickinson). Cells that were treatedwith Taxol (60 ng/ml) were harvested 24 h after treatment.

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1 This work was made possible by generous support from the Departmentof Medicine, Roger Williams Medical Center. L. E. S. was supportedin part by NIH Training Grant T32ES07272-05.